Construction Of The Arsenic Resistance Plasmids And The Conjugative Transfer To Acidithiobacillus Caldus

Bacterial leaching is the process using bacterial to accelate the sulfide minerals oxidation. Compared with chemical metallurgy, especially to the poor minerals, it has more advantages such as little pollution, little energy consume, low cost and so on. Now in our country, rich minerals become less and less, so it becomes much important to explore the poor minerals, but the conventional means has so many disadvantages, so bacterial leaching is fully applied to production. Bacterial leaching of sulfur-containing minerals in bioreactors has been commercialized in more than ten countries of the world at present.A variety of chemoautolithotrophic bacteria, which are capable of oxidizing iron-or sulfur-containing mineral can easily be isolated from acidic mine drainage and mineral ores. Traditionally, it was regarded that the most abundant microorganisms involved in the mineral-leaching process at mesophilic temperatures around 30 ℃ were Acidithiobacillus ferrooxidans (previously Thiobacillus ferrooxidans) and Acidithiobacillus thiooxidans (previously Thiobacillus thiooxidans). However from the continuous biooxidation reactors operating, it was established recently that at higher temperatures (between 40 and 50 ℃), Leptospirillum ferrooxidans and Acidithiobacillus caldus (previously Thiobacillus caldus) are the dominant organisms in oxidizing ores. Study has found that A. caldus can improve the efficiency of bioleaching when used together with iron-oxidizing bacteria of the genus of Leptospirillum and the role that A. caldus plays in the bioleaching process as bacterial consortia has been well documented.A. caldus was first characterized in 1994. It is a moderately thermophilic, extremely acidophilic, sulfur-oxidizing, chemolithotrophic bacterium, which canobtain energy from the chemolithotrophic oxidation of inorganic sulphur and its compounds and use this energy to support autotrophic growth on carbon dioxide. The cells are short, rod-shaped, motile, and Gram negative. The colonies formed on solid sodium thiosulfate medium are generally white, domed with regular margin, and transparent with a smooth surface. There is the deposition of sulfur in the center of the colonies. The optimal growth temperature was about 45 "C and the optimal pH was 2.0—2.5. From 32"C to 58*C, it can grow.In the recovery of gold from arsenopyrite ores, high concentration of arsenic killed the bacteria. So we have to use the genetic means to reconstruct the strains with high arsenical resistance. Since A. caldus was first characterized in 1994, the physiology of A. caldus has been well studied, but little information is available about its molecular genetics. The study about A. caldus in China started late.A moderately thermophilic sulfer-oxidizing bacterium MTH-04 was isolated in our lab from tengchong area, Yunnan Province in China. Based on the analysis of morphological, biochemical, physiological characters and 16S rDNA sequences, the acidophile, MTH-04 was considered another isolate of Acidithiobacillus caldus. In this experiment, using the recombinant technique in vitro, two new arsenic resistance plasmids pSDRA3 and pSDRA4 were constructed. By subcloning the arsenic resistance genes from plasmid pUM3 into the wide-host-range IncQ plasmid pMMB24 with the hybrid trp-lac (tad) promoter and mob site, a new plasmid pSDRA3 with arsenic resistance was constructed. Followed by deleting the regulative gene of the promoter, the lacfi gene, another arsenic resistance plasmid pSDRA4 was constructed. The genomic DNA of E. coli SM10 has the tra gene. So both pSDRA3 and pSDRA4 were transformed into E. coli SM10. Using E. coli SM10 (pSDRA3), E. coli SM10 (pSDRA4) as the donors, respectively, and A. caldus MTH-04 as the recipients, the plasmids could be mobilized into A. caldus strains with the aid of tra gene on the genomic DNA of E. coli SM10 with a frequency of (1.095 ±0.662) X10"5 and (1.444 + 0.797) X 10"4, respectively. A. caldus is an extremely acidophilic bacterium, which optimal pH for growth is 2.0-2.5, whereas E. coli is a heterotrophic bacterium whose optimal pH for growth is 7.0-7.5. Though there are markeddifferences in their growth conditions, the mating medium could provide energy sources for either E. coli or A. caldus. In order to test the transconjugant donors, another matings using A. caldus MTH-04 (pSDRA3), A. caldus MTH-04 (pSDRA4) as the donors, E. coli C600 as the recipients, E. coli C600 (RP4) as the assistant, was conducted on mating medium for 24 hours. The plasmids in the reverse-transconjugants were extracted and detected. The result showed that plasmids were really transformed from E. coli into A. caldus. Moreover all genetic markers of pSDRA3 and pSDRA4 were expressed in E. coli C600. The result showed that a genetic syetem between E. coli and A. caldus was really constructed.The stability of plasmids pSDRA3 and pSDRA4 in A. caldus MTH-04 was determined by checking for arsenic resistance. There are more than 81% and 76% of A. caldus MTH-04 cells, which carried the recombinant plasmids pSDRA3 or pSDRA4 respectively after being cultured for 50 generations without selective pressure, which showed that pSDRA3 and pSDRA4 were maintained consistently in A. caldus. Compared with wild type A. caldus, the level of the arsenic resistance of A. caldus (pSDRA3), A. caldus (pSDRA4) was respectively greatly raised from 10 mmol/Lto35mmol/Land 45mmoI/L.Our work not only first constructed the genetic transforming system between E. coli and A. caldus, open a way to investigate the gene expression mechanism and gene regulation in A. caldus, but also provide a new insight into how to improve the performance of A. caldus. The arsenic resistance strains A. caldus (pSDRA3) and A. caldus (pSDRA4) offered bioleaching strains as well.